Pigeon odor varies with experimental exposure to trace metal pollution

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Pigeon odor varies with experimental exposure to trace

metal pollution

Sarah Leclaire, Marion Chatelain, Anaïs Pessato, Bruno Buatois, Adrien

Frantz, Julien Gasparini

To cite this version:

Sarah Leclaire, Marion Chatelain, Anaïs Pessato, Bruno Buatois, Adrien Frantz, et al.. Pigeon odor varies with experimental exposure to trace metal pollution. Ecotoxicology, Springer Verlag, 2019, 28 (1), pp.76-85. �10.1007/s10646-018-2001-x�. �hal-02337484�

1

Pigeon odor varies with experimental exposure to trace metal pollution

2

Sarah LECLAIRE1,2,3, Marion CHATELAIN3,*, Anaïs PESSATO2,5,†, Bruno BUATOIS2,

3

Adrien FRANTZ3, Julien GASPARINI3

4

1

Laboratoire Evolution & Diversité Biologique, UMR 5174 (CNRS, Université Paul Sabatier,

5

ENFA), 118 route de Narbonne, 31062 Toulouse, France.

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2_{Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), UMR 5175, CNRS - Université de}

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Montpellier - Université Paul Valéry Montpellier - EPHE, 1919 route de Mende, 34293

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Montpellier, France

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3_{Sorbonne Université, UPEC, Paris 7, CNRS, INRA, IRD, Institut d’Ecologie et des Sciences}

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de l’Environnement de Paris, 75005 Paris, France

11

*

Current address: Wild Urban Evolution and Ecology Lab, Center of New Technologies,

12

University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland

13

†

Current address: Centre for Integrative Ecology, Deakin University, Geelong Waurn Ponds

14

Campus, Victoria 3217, Australia

15

Corresponding author: Sarah Leclaire, sarah.leclaire@free.fr; tel: +33 05 61 55 67 53;

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ORCID: 0000-0002-4579-5850

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18 19

2

Abstract

20

Trace metals are chemical pollutants that have well-known noxious effects on wildlife and

21

that are current major environmental issues in urban habitats. Previous studies have

22

demonstrated their negative (e.g. lead) or positive (e.g. zinc) effects on body condition,

23

immunity and reproductive success. Because of their effects on condition, trace metals are

24

likely to influence the production of condition-dependent ornaments. The last decade has

25

revealed that bird odors, like mammal odors, can convey information on individual quality

26

and might be used as secondary sexual ornaments. Here, we used solid-phase microextraction

27

headspace sampling with gas chromatography - mass spectrometry to investigate whether

28

plumage scent varied with experimental supplementation in lead and/or zinc in feral pigeons.

29

Zinc supplementation (alone or in combination with lead) changed the proportion of several

30

volatiles, including an increase in the proportion of hydroxy-esters. The production of these

31

esters, that most likely originate from preen gland secretions, may be costly and might thus be

32

reduced by stress induced by zinc deficiency. Although lead is known to negatively impact

33

pigeon condition, it did not statistically affect feather scent, despite most of the volatiles that

34

increased with zinc exposure tended to be decreased in lead-supplemented pigeons. Further

35

studies should evaluate the functions of plumage volatiles to predict how trace metals can

36

impact bird fitness.

37 38

Keywords: Scent; Zinc; Lead; Birds; Dove

3

Introduction

40

Trace metals (e.g. lead, iron, copper, zinc) are normally present in very low levels in the

41

environment and small amounts are essential part of physiology in animals and plants (Powell

42

2000, Prasad 2009, Plum et al. 2010). For instance, zinc is essential in regulating homeostasis,

43

immune responses, and oxidative stress (Stefanidou et al. 2006), and zinc deficiency has

44

serious consequences that have been recognized for many years and that include oxidative

45

damages, diabetes and congenital malformations (Fosmire 1990; Oteiza et al. 1995; Prasad

46

2003). However, excessive quantities of trace metals can be toxic. The large amount of trace

47

metals that have been released in the environment by anthropogenic activities (Nriagu 1979)

48

have well-known noxious effects on humans (Järup 2003; Tchounwou et al. 2012) and

49

wildlife (Demayo et al. 1982; Goutte et al. 2014; Scheuhammer et al. 2007). The binding

50

potential (Godwin 2001; Tainer et al. 1992) and the propensity of trace metals to catalyze

51

oxidation reactions (Koivula and Eeva 2010) are responsible for immunosuppression

52

(Chatelain et al. 2016c; Snoeijs et al. 2005; Snoeijs et al. 2004), oxidative stress (Berglund et

53

al. 2007) and endocrine disruptive effects (Baos et al. 2006; Meillère et al. 2016). For

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instance, lead, whose widespread use has caused extensive environmental contamination

55

(Nriagu 1996), is associated with impaired learning in herring gulls (Larus argentatus)

56

(Burger and Gochfeld 2005), reduced sperm quality in red deer (Cervus elaphus) (Castellanos

57

et al. 2015), and higher oxidative stress in Mozambique tilapias (Oreochromis mossambicus)

58

(Kaya and Akbulut 2015).

59

Sexual ornaments are conspicuous phenotypic traits that can take various forms such

60

as large antlers, bright plumage, or elaborated song, and that are displayed to attract mates.

61

Because their expression can be influenced by genotype and environment, including various

62

stress factors, such as food availability, extreme temperatures, noise and parasites (McGraw et

63

al. 2002; Troïanowski et al. 2017; von Schantz et al. 1999), they often honestly reveal the

4

quality of the bearer (Zahavi and Zahavi 1999). By inducing physiological stress, trace metals

65

can also alter ornament expression, as shown for the plumage coloration and singing

66

behaviour of birds (Chatelain et al. 2017; Giraudeau et al. 2015; Gorissen et al. 2005;

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allverd -Coll et al. 2015). Odor cues are also subjected to physiological and environmental

68

influences (Ferkin et al. 1997; Johnston and Bronson 1982; Penn and Potts 1998), and are

69

major secondary sexual traits in a wide range of species (Blaustein 1981; Havlicek et al. 2005;

70

Martín and López 2006; Wyatt 2014). However, the effects of environmental pollution on

71

their expression are largely unknown.

72

In this study, we tested the hypothesis of the impact of trace metals exposure (zinc and

73

lead) on plumage odor in feral pigeon (Columbia livia) by an experimental supplementation

74

approach. Although birds have long been considered to rely primarily on visual and acoustic

75

cues, the past decade has revealed that birds use olfaction in various contexts including mate

76

choice or parental care (reviewed in Caro et al. 2015). Bird odors, like mammal odors, can

77

vary with genotype (Leclaire et al. 2014b), sex-hormone levels (Whittaker et al. 2017) and

78

diet (Thomas et al. 2010), and are mainly composed of alkanes, alkanols, esters and fatty

79

acids (reviewed in Campagna et al. 2012) that may be energetically costly to produce. Using a

80

similar experimental design, we have already described beneficial effects of zinc on body

81

mass, immunity and female investment into eggs (Chatelain et al. 2016b; Chatelain et al.

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2016c), and detrimental effects of lead on iridescent coloration, the maternal transfer of

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antibodies into eggs and nestling growth (Chatelain et al. 2016b; Chatelain et al. 2017). We

84

predict therefore that if pigeon odor is condition-dependent, it should vary in opposite

85

direction with experimental exposure to lead and zinc.

86 87

Material and methods

88

Capture and housing

5

From February 20 to March 27 2013, we captured 96 free-living adult feral pigeons in several

90

locations in Paris. We immediately transferred them in 8 outdoor aviaries (2.20m x 2.20m) at

91

the CEREEP field station (Centre d’Ecologie Expérimentale et Prédictive - Ecotron

Ile-de-92

France, UMS 3194, Ecole Normal Supérieure, Saint-Pierre-lès-Nemours, France).

93 94

Metal supplementation

95

This study is part of a bigger study investigating the effect of zinc and lead on several life

96

history traits of pigeons. Details on the protocol have thus been published elsewhere

97

(Chatelain et al. 2016b; Chatelain et al. 2016c; Chatelain et al. 2017). Briefly, on April 8

98

2013, after an acclimation period, aviaries were divided into 4 metal treatments (2 aviaries per

99

treatment). Birds were evenly distributed among aviaries according to their flock, sex and

100

plumage eumelanin level (Chatelain et al. 2016b). Treatments were lead supplementation (tap

101

water enriched with 1 ppm lead acetate), zinc supplementation (tap water enriched with 10

102

ppm zinc sulphate), lead and zinc supplementation (tap water enriched with 1 ppm lead

103

acetate and 10 ppm zinc sulphate) and no supplementation, i.e. control (tap water with no

104

metal added). We chose these concentrations based on the lead concentration measured in

105

blood of urban birds (Roux and Marra 2007) and on the gastrointestinal absorption rate of

106

lead in zebra finches (Dauwe et al. 2002). Zinc concentrations were approximated using the

107

zinc/lead concentration ratio measured in the environment and in bird feathers (on average,

108

zinc is 10 times more concentrated than lead; Azimi et al. 2005; Chatelain et al. 2014; Frantz

109

et al. 2012). Drinking bowls and baths were filled every other day with treated water. The

110

efficiency of our supplementation protocol to increase bird exposure to lead and zinc has been

111

previously validated (Chatelain et al. 2016b).

112 113

Sample collection

6

After 20 weeks of treatment, a few feathers (~33 mg) were cut in the lower back region of 74

115

birds (37 males and 37 females), and stored in 4 ml PTFEfaced septum sealed glass vials at

-116

80°C until analysis. Birds from five aviaries (two lead-supplemented aviaries, one

zinc-117

supplemented aviary, one zinc-and-lead-supplemented aviary and one control aviary) were

118

sampled on 27 August 2013, while birds from the remaining three aviaries (one

zinc-119

supplemented, one zinc-and-lead supplemented and one control aviaries) were sampled on the

120

next day. The 74 birds sampled included 20 control birds, 19 lead-supplemented birds, 17

121

zinc-supplemented birds and 18 zinc-and-lead-supplemented birds.

122 123

Chemical analyses

124

We extracted chemical compounds with solid phase microextraction (SPME) using a

125

stableflex 50/30μm D B (divinylbenzene) / CAR (carboxen) / PDMS (polydimethylsiloxan)

126

fiber (Supelco, Sigma-Aldrich, Bellefonte, PA, USA). To clear headspace for SPME

127

extraction, we packed feathers at the bottom of the vial, using a glass rod cleaned with

128

dichloromethane and ethanol between samples. The sample was then placed for 20 min in a

129

laboratory incubator maintained at 34°C. We chose 34°C to mimic the natural temperature of

130

bird body surface during a Parisian summer. During the summer 2013, the average daily

131

maximal temperature in Paris was 25.3°C (public data of Meteo France). At 25°C ambient

132

temperature and low humidity, surface temperature of birds is 33.4°C (Chilgren and King

133

1973). The SPME fiber was then exposed to the headspace (without touching the feathers) for

134

25 minutes at 34°C, after which the fiber was retracted and the adsorbed chemicals were

135

analyzed by gas chromatography-mass spectrometry (GC-MS). Only one SPME fiber was

136

used in the study.

137

We analyzed the samples using a Shimadzu GCMS-QP2010plus (Shimadzu Scientific

138

Instruments, Kyoto, Japan) equipped with an Optima®-5MS column (30 m x 0.25 mm x 0.25

7

μm, Macherey-Nagel, Düren, Germany) at the Plateforme d’Analyses Chimiques en Ecologie

140

(PACE), technical facilities of the LABoratoire d’Excellence named Centre Méditerranéen de

141

l’Environnement et de la Biodiversité (CEMEB). Helium was used as the carrier gas, at a

142

constant 1ml/min flow rate. Fibers were exposed in a 200 °C injection port to desorb volatiles.

143

We ran the following temperature protocol: 40° for 5 min, 40°C to 180°C ramped at 5°C/min,

144

180°C to 250°C ramped at 10°C/min, and finally held at 250°C for 1 min. The ion source and

145

transfer line temperatures was 200°C and 250°C respectively. Mass were scanned from 38 -

146

350 m/z in electron ionization mode. Fibers were removed from the injection port after 10

147

min of desorption. We regularly interspersed blanks throughout the sample analyses to detect

148

contamination. We ran an alkane standard solution (Alkanes standard solution, 04070, Sigma

149

Aldrich®, Germany) under the same conditions for retention index calculation. The samples

150

from 9 controls, 9 lead-supplemented pigeons, 10 zinc-supplemented birds and 11

zinc-and-151

lead-supplemented birds were ran in March 2015, while the other samples were ran in June

152

2015. Within each analysis time period (March vs. June 2015), samples were ran randomly.

153

Consequently, there was no difference in date of analysis between zinc-supplemented and

154

non-zinc supplemented samples or between lead-supplemented and non-lead supplemented

155

samples (all P > 0.15). Ten samples had very low GCMS signal and were not included in the

156

statistical analyses.

157

To confirm that the feather odor captured from captive pigeons was representative of

158

that of wild pigeons, we compared the chromatograms with those of two samples of back

159

feathers collected just after capture in February 2013. In addition, to shed light on the

160

potential origin of plumage chemicals, we compared the feather profiles to those of preen

161

secretion samples collected on two pigeons just after capture.

162

Chromatograms were analyzed blind to treatment using the Shimadzu GC-MS

163

Solution software. Analytes were identified by comparison of mass spectral data using the

8

NIST library 2011 and cross-checking linear retention index of the spectral match found in

165

the literature (PubChem database) with the calculated linear retention index of the analytes.

166

Four monoterpenes were detected in pigeon feathers (α-Pinene, β-Pinene, p-Cymene and

167

Bornyl acetate; Table 1). Most probably of plant origin (Charpentier et al. 2012), they were

168

excluded from the analyses. One peak corresponded to a co-elution of several compounds

169

including one contaminant (Table 1), and it was also not included in the analyses.

170

To ascertain that our GCMS analyses were repeatable, 5 samples were analyzed twice

171

with a few days interval (range: 5 - 8 days). Results show that total chromatogram area was

172

lower in the second analysis than in the first analysis (paired t-test: t4 = 5.32, P = 0.006) but

173

that the composition of the two samples from the same bird was repeatable (PERMANOVA

174

analyses with bird identity as fixed factor: F4,4 = 8.93, P = 0.001).

175 176

Statistical analyses

177

Because, for most samples, we did not control for the amount of feathers collected,

178

and because an internal standard was not included in the GCMS analyses, we did not rely on

179

the absolute abundance of chromatogram peaks for the analyses of chemical composition;

180

rather, we quantified each peak as the proportion of the peak size relative to the total area of

181

the chromatogram. Proportions of chemical compounds were then transformed using

182

isometric log ratio "ilr" (Brückner and Heethoff 2017). In addition, to limit the effect of the

183

differences in the amount of feathers on odor composition, we restricted the odor composition

184

analyses on the peaks that each represented on average more than 0.75% of the chromatogram

185

(n = 19 peaks representing on average 91.0 % ± 0.3 % of the total chromatogram areas, Table

186

1). All of these peaks, except one, were present in all birds.

187

To determine if the chemical composition of plumage odor was related to lead and

188

zinc exposition, we used a PERMANOVA with 5000 permutations i.e. nonparametric

9

multivariate analysis of variance, (Adonis2 function, "vegan" package in R; Oksanen et al.

190

2013), based on Euclidean distance. The two-way interaction between lead and zinc, the bird

191

sex and the analysis time period (March vs. June 2015) were also included in the model. The

192

date of feather sampling was used as strata, thus restricting permutations within date of

193

sampling. In Adonis, significant differences may be caused by different within-group

194

dispersion instead of different mean values of the groups (Warton et al. 2012). For significant

195

effects, we therefore tested difference in dispersion between groups (Betadisper function,

196

"vegan" package; Oksanen et al. 2013).

197

To determine the chemical compounds that were preferentially associated with a

198

significant fixed effect, we used correlation indices (function "multipatt" in the "indicspecies"

199

package in R; De Caceres et al. 2016) with the point biserial coefficient of association on

200

proportions of chemical compounds (De Cáceres and Legendre 2009). We restricted

201

permutations within date of sampling. This method uses permutation tests to test significance

202

of value, but does not correct for multiple tests. Multiple testing corrections are flawed

203

because they assume multiple tests are independent, whereas multiple volatiles can show

204

correlated effects (Fig. S1) because of the nature of the data (relative abundance vs. absolute

205

abundance) and because the biosynthesis pathway of these volatiles may be related.

206

Therefore, we also performed robust principal component analyses (RPCA) on the

ilr-207

transformed dataset (Filzmoser et al. 2009) to identify the subset of compounds that varied

208

with treatment. RPCA axis scores were compared between zinc-supplemented and non-zinc

209

supplemented birds using linear mixed models with zinc treatment and analysis time period as

210

fixed effects, and date of feather sampling as a random factor.

211

We performed all statistical analyses using the R statistical software (R Core Team

212

2017) and used a significance level set at α = 0.05.

213 214

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Results

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We detected a total of 45 peaks in the 63 odor profiles of pigeons, that included alkanes,

216

aldehydes, alcohols, ketones, and esters (Table 1 and Fig. 1). Only 6 (13%) of these

217

compounds were also detected in preen secretions (Table 1). In contrast, 39 of them (87%)

218

were also detected in the plumage of wild Parisian pigeons, suggesting that captivity had only

219

slight influence on the identity of feather volatiles.

220

Chemical composition varied with zinc supplementation (F1,61 = 4.27, P =

221

0.016; Fig. 2 and Fig. S2), but not with lead supplementation (F1,60 = 0.62, P = 0.89) or the

222

interaction between zinc and lead (F1,59 = 1.22, P = 0.56). It did not vary with sex (F1,60 = 0.14,

223

P = 0.99), but varied with the period when the samples were analyzed (F1,61 = 13.20, P <

224

0.001). Multivariate dispersion in chemical composition between zinc-supplemented and

non-225

zinc supplemented birds was similar (F1,62 = 0.59, P = 0.45). Correlation indices showed that

226

the proportions of the two hydroxy esters and 3-Hexanone were increased in zinc

227

supplemented birds, while the proportion of Nonanal was reduced (Fig. 3). Similarly, a robust

228

PCA showed that the second principal component, which accounted for 26% of the variance

229

and which was positively related to the two hydroxy esters and the two ketones, was higher in

230

zinc-supplemented birds than in non-zinc supplemented birds (χ²1 = 7.14, P = 0.008; Fig. 4).

231

232

Discussion

233

By analyzing feather volatiles of feral pigeons chronically exposed to zinc and/or lead, we

234

provide evidence for an effect of zinc on pigeon odor composition. In particular, we found

235

that zinc mainly increased the relative abundance of the two hydroxy esters and 3-Hexanone.

236

All the compounds affected by zinc supplementation may potentially be produced

237

endogenously by the bird. However, in contrast to several species, where most plumage

11

chemicals originate from the preen gland (Leclaire et al. 2011; Mardon et al. 2011; Zhang et

239

al. 2009), only the two hydroxy-esters were detected in pigeons' preen secretions. Although

240

the sample size used to study the difference between preen secretion and feather volatiles was

241

low (n = 2 birds sampled for preen secretions), this result is consistent with a study on

242

common wood pigeons (Columba palumbus), where 93% of the whole plumage lipids do not

243

originate from preen secretions (Jacob and Grimmer 1975).

244

The other compounds affected by zinc exposure were not detected in preen secretions,

245

and may rather originate from other lipid-producing areas in the integument (Jacob and

246

Ziswiler 1982) or from feather bacteria metabolism (Whittaker and Theis 2016). The role of

247

bacteria in animal olfactory signaling has recently received renewed interests (Archie and

248

Theis 2011; Douglas and Dobson 2013; Ezenwa and Williams 2014) and a few experimental

249

studies in mice, elephants (Loxodonta africana), Indian mongooses (Herpestes

250

auropunctatus) and European hoopoes (Upupa epops) have shown that some major odorants

251

require commensal bacteria for their production (Goodwin et al. 2016; Gorman et al. 1974; Li

252

et al. 2013; Martin-Vivaldi et al. 2010). Methyl-branched alkanes, methyl ketones, and

253

aldehydes can all be released by bacterial degradation of fatty acids (Schulz and Dickschat

254

2007). Feathers of zinc-supplemented pigeons have lower bacterial load and different

255

bacterial assemblage than feathers of birds non-exposed to zinc (Chatelain et al. 2016a),

256

which may have led to differences in the composition of feather volatiles between birds.

257

Further studies on the mechanisms of avian odor production are clearly needed to understand

258

how environment can affect feather volatiles.

259

We found that zinc increased the proportion of preen-gland-related compounds, which

260

might suggest higher production of preen oil in zinc-exposed birds. Several studies have

261

suggested that birds in higher condition are able to produce higher quantity of preen oil. For

262

instance, experimental infections and immune challenge reduce the size of the preen gland in

12

house sparrows (Passer domesticus) and tawny owls (Strix aluco) (Moreno‐Rueda 2015;

264

Pap et al. 2013; Piault et al. 2008). Zinc provided at low dose is essential for many biological

265

processes (Stefanidou et al. 2006), and accordingly, zinc supplementation has various

266

beneficial effects on pigeon condition (Chatelain et al. 2016b; Chatelain et al. 2016c).

267

Therefore, zinc, through its positive effects on bird condition, may increased the ability of

268

bird to produce wax esters and other costly volatiles. Although avian odors have reapeatedly

269

been shown to vary with genetics (i.e., Krause et al. 2012; Leclaire et al. 2017) and

270

physiological status (Douglas et al. 2008; Whittaker et al. 2018), a very few studies have

271

shown the effect of environmental stressors on their expression (Bombail et al. 2018). For

272

instance, infection influences the wax ester profiles of preen oil in song sparrows (Melospiza

273

melodia) and the volatile profiles of feces in mallards (Anas platyrhynchos) (Grieves et al.

274

2018; Kimball et al. 2013). Our study, which shows that the volatile profiles of feather is

275

related to zinc levels in the environment, is therefore consistent with the stress-induced effects

276

on odors demonstrated in other birds.

277

In several avian species, odor plays a crucial role in social interactions (Balthazart and

278

Schoffeniels 1979; Balthazart and Taziaux 2009; Hirao et al. 2009). Zinc-induced changes in

279

pigeon scent might therefore influence the behavior of conspecifics, with, for instance,

280

females avoiding the scent of zinc-deficient males. However, although pigeons are

well-281

known to use their olfactory abilities for homing (reviewed in Wallraff 2005), it is unknown

282

whether they use also olfaction for social communication. Feather chemicals may play also a

283

role in social communication by affecting feather coloration or condition (Moreno‐Rueda

284

2017). For instance, in house finches (Carpodacus mexicanus), preen oil enhances the

285

coloration of red feathers (López-Rull et al. 2010). In pigeons, changes in the coloration of

286

melanic feathers have been induced by zinc supplementation (Chatelain et al. 2017), and

287

might therefore be mediated by changes in the chemical composition of feather volatiles.

13

Feather chemicals do not only play a role in social communication; they can protect

289

feathers from wear and act as chemical defenses against parasites (Douglas 2013; Hagelin and

290

Jones 2007). For instance, in rock pigeons, preen oil has positive effects on plumage condition

291

(Moyer et al. 2003). 3-Hexanone, which was increased in zinc-exposed birds, is a common

292

urine component of mammals, including humans (Burger et al. 2006; delBarco‐Trillo et al.

293

2011; Raymer et al. 1986; Smith et al. 2008), and has been detected in the feathers of white

294

Leghorn chickens (Gallus gallus domesticus L.) (Bernier et al. 2008) and king penguins

295

(Gabirot et al. 2018). It belongs to the volatile methyl-ketone family, whose some compounds

296

can provide protection against parasites (Borges et al. 2015; Kirillov et al. 2017). According

297

to the parasite-repellent function of volatile ketones, volatile methyl-ketones of dark-eyed

298

juncos (Junco hyemalis) are increased during the breeding season (Soini et al. 2007), when

299

the need for protection against nest parasites is high. Zinc, by inducing higher production of

300

hydroxy-esters and 3-hexanone, might thus enhance plumage condition and the anti-parasite

301

properties of feathers, thus adding to the numerous positive effects of zinc on pigeon

302

(Chatelain et al. 2016b; Chatelain et al. 2016c).

303

Lead is a toxic trace metal that can impair various biological processes, leading to

304

illness and mortality (Papanikolaou et al. 2005). In feral pigeons, lead decreases iridescent

305

coloration, the maternal transfer of antibodies into eggs, and nestling growth (Chatelain et al.

306

2016b; Chatelain et al. 2017). Despite these detrimental effects on pigeon condition, we did

307

not detect effects of lead on pigeon scent. As our experimental exposure to lead

308

underestimated the natural range (lead was 1.5 times less concentrated in the feathers of

lead-309

supplemented pigeons than in those of wild feral pigeons; Chatelain et al. 2016b), we cannot

310

exclude that, in urban areas, the high concentration of lead negatively impacts odor

311

production in birds.

14

We did not find evidence for a sex signature in the feather odor of wild-caught pigeons

313

kept in captivity. Similarly, no sex-differences in preen secretion composition have been

314

detected in non-breeding rock pigeons (Montalti et al. 2005). Although sexual dimorphism in

315

chemical cues from feathers or preen secretions has been reported in several bird species

316

(Amo et al. 2012; Leclaire et al. 2011; Whittaker et al. 2010; Zhang et al. 2010), it is not

317

ubiquitous. For instance, king penguins (Aptenodites patagonicus) and Cory's shearwaters

318

(Calonectris borealis) seem to exhibit no sex-differences in scent (Gabirot et al. 2018;

319

Gabirot et al. 2016). Pigeons display sexual polymorphism in iridescent coloration of neck

320

feathers (Chatelain et al. 2017; Leclaire et al. 2014a), and might, therefore, use this other trait

321

to discriminate between sexes. Alternatively, because the molecules detected using GC-MS

322

depend on the analysis strategy (e.g., extraction protocol and GC column), the profile of

323

chemical composition presented here is not exhaustive. For instance, carboxylic acids, which

324

are better resolved using a polar column (Whelan et al. 2010), might encode sex information.

325

In conclusion, our study showed that exposure to a moderate amount of zinc induced

326

changes in the feather volatile composition of feral pigeons. Future studies should now

327

evaluate the role of these volatiles in social communication, protection against parasites or

328

feather wear to further predict how trace metals, which are chemical pollutants of prime

329

concern in cities, can impact bird fitness.

330

331

Acknowledgements

332

We thank the "Mairie de Paris" (Thomas Charachon) for allowing the capture of birds and the

333

Centre de Recherche en Ecologie Experimentale et Predictive (CEREEP) which provided

334

logistic support for the field work of this study. We are very thankful to T. Gayet, S. Pollet, S.

335

Hasnaoui, F. Lorente, S. Perret and B. Decenciere for their help in field work.

15 337

Funding

338

This work was financed by grants from the local government (Ile-de-France: Sustainable

339

Development Network R2DS, No. 2012–11 to J.G.), and from the "Agence Nationale de la

340

Recherche" (No. ANR-13-PDOC-0002 to S.L.).

341 342

Conflict of interest

343

The authors declare that they have no conflict of interest.

344

345

Compliance with Ethical Standards

346

All experiments were carried out in strict accordance with the recommendations of the

347

"European Convention for the Protection of vertebrate Animals used for Experimental and

348

Other Scientific Purposes" and were conducted under the authorizations of the "Ministère de

349

l’éducation nationale, de l’enseignement supérieur et de la recherche” (authorization

350

N_00093.02) and the "Direction Departementale des Services Veterinaires de

Seine-et-351

Marne" (authorization N_ 77-05). The authors declare no conflict of interest.

352 353

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607 608

21

Figure captions

609

Figure 1: Representative chromatogram of feather volatiles in captive pigeons. Letters refer

610

to compounds listed in table 1.

611 612

Figure 2: Partial unconstrained distance-based redundancy analysis plot showing separation

613

between pigeons by zinc supplementation. The effect of the period when the samples were ran

614

on the GCMS was partialled out to better show separation.

615 616

Figure 3: Mean ± SE proportion of the four chemical compounds that varied with zinc

617

exposure in zinc-supplemented birds and non zinc-supplemented birds. P values were

618

calculated using a mixed model with date of sampling as a random effect and analyses time

619

period as covariate.

620 621

Figure 4: Robust principal component analysis plot showing separation between pigeons by

622

zinc supplementation. Vectors representing chemical compounds with high loading on axis 2

623

are highlighted.

624 625 626

22

Table 1: Chemical compounds putatively identified in the feathers of feral pigeons.

627

Compounds in bold are those that were > 0.75 % of the total chromatogram and that were

628

used in statistical analyses.

629 630 Name Letter (see figure 1) Retention index calculated Detected in preen secretions Detected on the plumage of wild Parisian pigeons Ketones Hexan-3-one a n.d. x 6-methyl-5-Hepten-2-one b 982 x Aldehydes Hexanal c n.d. x Heptanal d 896 x Nonanal e 1101 x x Decanal 1202 x x Alkanes Octane f n.d. x 2, 4-dimethyl-Heptane 817 x Nonane g 899 x Branched alkane #1 h 1060 x Branched alkane #2 o 1065 x Branched alkane #3 i 1104 x Branched alkane #4 1109 x Dodecane 1200 x

23 Branched alkane #5 1277 x Branched alkane #6 j 1283 x Branched alkane #7 1305 x Branched alkane #8 1322 x Branched alkane #9 k 1329 x Branched alkane #10 1337 x Branched alkane #11 1345 x Branched alkane #12 1348 x Tridecane 1300 x Pentadecane 1500 x Alcohols Pentanol n.d. x 2-butoxy-Ethanol 903 x 1-butoxy-2-Propanol 940 x 1-Octen-3-ol 978 x 2-ethyl-Hexan-1-ol l 1028 x 2, 6-dimethyl-7-Octen-2-ol 1071 x Esters

Butanoic acid, methyl ester n.d. x

Propanoic acid, propyl ester 804

Acetic acid, butyl ester m 809 x

Hexanoic acid, methyl ester 922 x

Hydroxy ester #1 n 1357 x x

24 Fatty acids Nonanoic acid 1273 x Unknown Unknown #1 p 884 x Unknown #2 q 910 Unknown #4 1143 Unknown #5 1232 Unknown #6 r 1295 Unknown #7 1307 x Unknown #8 1316 2, 2, 4, 6, 6-pentamethylheptane + hexanoic acid a s 993 x x n.d.: not determined a

: These two compounds were eluted together.

631 632